Methods for the modulation of the growth of collateral arteries and/or other arteries from preexisting arteriolar connections

ABSTRACT

Described is the modulation of the growth of collateral arteries and/or other arteries from preexisting arteriolar connections. Methods are provided for enhancing the growth of collateral arteries and/or other arteries from preexisting arteriolar connections comprising contacting tissue or cells with a monocyte chemotactic protein (MCP) or a nucleic acid molecule encoding said MCP. Furthermore, the use of a MCP or a nucleic acid molecule encoding said MCP for the preparation of pharmaceutical compositions for enhancing collateral growth of collateral arteries and/or other arteries from preexisting arteriolar connections is described. Also provided are methods for the treatment of tumors comprising contacting tissue or cells with an agent which suppresses the growth of collateral arteries and/or other arteries from preexisting arteriolar connections through the attraction of monocytes. Described is further the use of an agent which suppresses the growth of collateral arteries and/or other arteries from preexisting arteriolar connections through attraction of monocytes for the preparation of pharmaceutical compositions for the treatment of tumors.

[0001] The present invention relates generally to the modulation of thegrowth of collateral arteries or other arteries from preexistingarteriolar connections. In particular, the present invention provides amethod for enhancing the growth of collateral arteries and/or otherarteries from preexisting arteriolar connections comprising contactingtissue or cells with a monocyte chemotactic protein (MCP) or a nucleicacid molecule encoding said MCP. The present invention also relates tothe use of an MCP or a nucleic acid molecule encoding said MCP for thepreparation of pharmaceutical compositions for enhancing collateralgrowth of collateral arteries and/or other arteries from preexistingarteriolar connections. Furthermore, the present invention relates to amethod for the treatment of tumors comprising contacting tissue or cellswith an agent which suppresses the growth of collateral arteries and/orother arteries from preexisting arteriolar connections through theattraction of monocytes. The present invention further involves the useof an agent which suppresses the growth of collateral arteries and/orother arteries from preexisting arteriolar connections through theattraction of monocytes for the preparation of pharmaceuticalcompositions for the treatment of tumors.

[0002] In the treatment of subjects with arterial occlusive diseasesmost of the current treatment strategies aim at ameliorating theireffects. The only curative approaches involve angioplasty (balloondilatation) or bypassing surgery. The former carries a high risk ofrestenosis and can only be performed in certain arterial occlusivediseases, like ischemic heart disease. The latter is invasive and alsorestricted to certain kinds of arterial occlusive diseases. There is noestablished treatment for the enhancement of collateral growth.

[0003] Vascular growth in adult organisms proceeds via two distinctmechanisms, sprouting of capillaries (angiogenesis) and in situenlargement of preexisting arteriolar connections into true collateralarteries¹. Recent studies have disclosed mechanisms leading toangiogenesis with vascular endothelial growth factor (VEGF) as a majorcomponent²⁻⁶. This specific endothelial mitogen is upregulated byhypoxia and is able to promote vessel growth when infused into rabbithindlimbs after femoral artery excision^(7,8). These studies however didnot distinguish between capillary sprouting, a mechanism calledangiogenesis, and true collateral artery growth. Whereas VEGF is onlymitogenic for endothelial cells, collateral artery growth requires theproliferation of endothelial and smooth muscle cells and pronouncedremodeling processes occur^(1,9-12). Furthermore mainly capillarysprouting is observed in ischemic territories for example in the pigheart or in rapidly growing tumors^(1,3,13,14). True collateral arterygrowth, however, is temporally and spacially dissociated from ischemiain most models studied^(1,15). Other or additional mechanisms as thosedescribed for angiogenesis in ischemic territories are therefore neededto explain collateral artery growth. From previous studies it is knownthat these collateral arteries grow from preexisting arteriolarconnections¹.

[0004] However, while agents such as VEGF and other growth factors arepresently being employed to stimulate the development of angiogenesisafter arterial occlusion, such agents are not envisaged as being capableof modulating the growth of preexisting arteriolar connections into truecollateral arteries.

[0005] Thus, the technical problem of the present invention is toprovide pharmaceutical compositions and methods for the modulation ofthe growth of collateral arteries and/or other arteries from preexistingarteriolar connections.

[0006] The solution to this technical problem is achieved by providingthe embodiments characterized in the claims.

[0007] Accordingly, the invention relates to a method for enhancing thegrowth of collateral arteries and/or other arteries from preexistingarteriolar connections comprising contacting tissue or cells with amonocyte chemotactic protein (MCP) or a nucleic acid molecule encodingsaid MCP.

[0008] For the purpose of the present invention the growth of arteriesfrom preexisting arteriolar connections is also called “arteriogenesis”.In particular, “arteriogenesis” is the in situ growth of arteries byproliferation of endothelial and smooth muscle cells from preexistingarteriolar connections supplying blood to ischemic tissue, tumor orsites of inflammation. These vessels largely grow outside the affectedtissue but are much more important for the delivery of nutrients to theischemic territory, the tumor or the site of inflammation thancapillaries sprouting in the diseased tissue by angiogenic processes.

[0009] In the context of the present invention the term “monocytechemotactic protein” or “MCP” refers to proteins and peptides which canact on monocytes and lead to augmentation of monocyte activationaccumulation and migration³⁵. Thus, according to the present invention,any MCP or other substances which are functionally equivalent to an MCP,namely which are capable of activating and attracting monocytes can beused for the purpose of the present invention. The action of the MCPsemployed in the present invention may not be limited to theabove-described specificity but they may also act on, for exampleeosinophils, lymphocyte subpopulations and/or stem cells.

[0010] In accordance with the present invention, it has been found thatthrough the attraction of monocytes by monocyte chemotactic protein-1(MCP-1) the growth of collateral arteries and arteriogenesis could besignificantly enhanced in animals after femoral artery occlusion.Experiments performed within the scope of the present inventiondemonstrate that local infusion of MCP-1 increases both collateral—andperipheral conductance after femoral artery occlusion due to enhancedvessel growth by augmentation of monocyte accumulation concomitant withproliferative effects on endothelial and/or smooth muscle cells. Thus,MCPs or nucleic acid molecules encoding MCPs can be used to attractmonocytes to a certain tissue or cell which in turn leads to growth ofcollateral arteries as well as to growth of arteries from preexistingarteriolar connections, which is needed for the cure of severalocclusive diseases.

[0011] MCP-1 is a 14-kDa glycoprotein secreted by many cells, includingvascular smooth muscle- and endothelial cells²⁹⁻³² and induces monocytechemotaxis at subnanomolar concentrations³³. MCP-1 is a potent agonistfor the β chemokine receptors CCR 2 and CCR 4 which are both mainlyexpressed by monocytes but also have been found to be present onbasophils, T- and B-lymphocytes³⁴. These G-protein coupledseven-transmembrane-domain receptors lead to the activation of monocytesand increased adhesiveness of integrins, a process which finally leadsto monocyte arrest on endothelial cells³⁵. The MCP-1 gene shows largeinterspecies homologies³⁰ and can be induced by various cytokines (e.g.Tumor necrosis factor α) and immunoglobulin G³⁶. Recently it has beenshown in vitro that gene expression and protein secretion of MCP-1 arealso upregulated by shear stress and cyclic strain¹⁶⁻¹⁸. Thesemechanical forces have recently been shown to increase monocytechemotactic protein-1 (MCP-1) secretion in cultured human endothelialcells leading to increased monocyte adhesion¹⁶⁻¹⁸. These findingscomplement the observation that monocytes adhere and migrate into thevessel wall of collateral arteries after induction of coronary arterystenosis in the dog heart at a time when the proliferation index ismaximally increased¹⁹. Furthermore, monocyte accumulation is alsoobserved in the pig microembolization model of angiogenesis²⁰. Moreoverincreased levels of MCP-1 mRNA were found in ischemic tissue ofmicroembolized porcine myocardium²¹ as well as in reperfused ischemicmyocardium³⁷. However, although there are several reports published thatindicate that monocytes are involved in angiogenesis²²⁻²⁴ monocytes werenot believed to play a role in the development of collateral arteriesand arteriogenesis²⁵.

[0012] The MCPs to be employed in the methods and uses of the presentinvention may be obtained from various sources described in the priorart; see, e.g., Proösl^(69,) Dahinden⁷⁰, Alam⁷¹ and Oppenheim⁷². Thepotential exists, in the use of recombinant DNA technology, for thepreparation of various derivatives of MCPs comprising a functional partthereof or proteins which are functionally equivalent to MCPs asdescribed above. In this context, as used throughout this specification“functional equivalent or “functional part” of an MCP means a proteinhaving part or all of the primary structural conformation of an MCPpossessing at least the biological property of attracting monocytes. Thefunctional part of said protein or the functionally equivalent proteinmay be a derivative of an MCP by way of amino acid deletion(s),substitution(s), insertion(s), addition(s) and/or replacement(s) of theamino acid sequence, for example by means of site directed mutagenesisof the underlying DNA. Recombinant DNA technology is well known to thoseskilled in the art and described, for example, in Sambrook et al.(Molecular cloning; A Laboratory Manual, Second Edition, Cold SpringHarbour Laboratory Press, Cold Spring Harbour N.Y. (1989)). For example,it was found that a mutation of the amino acids Leu25 and Val27 into Tyrintroduces a novel monocyte chemoattractant activity into intedeukin-8,which normally does not activate monocytes⁶⁶.

[0013] MCPs or functional parts thereof or proteins which arefunctionally equivalent to MCPs, may be produced by known conventionalchemical syntheses or recombinant techniques employing the amino acidand DNA sequences described in the prior art⁶⁹⁻⁷², for example, MCPs maybe produced by culturing a suitable cell or cell line which has beentransformed with a DNA sequence encoding upon expression under thecontrol of regulatory sequences an MCP or a functional part thereof or aprotein which is functionally equivalent to MCP. Suitable techniques forthe production of recombinant proteins are described in, e.g., Sambrook,supra. Methods for constructing MCPs and proteins as described aboveuseful in the methods and uses of the present invention by chemicalsynthetic means are also known to those of skill in the art.

[0014] In another embodiment, the invention relates to the use of amonocyte chemotactic protein (MCP) or a nucleic acid molecule encodingsaid MCP for the preparation of a pharmaceutical composition forenhancing collateral growth of collateral arteries and/or other arteriesfrom preexisting arteriolar connections.

[0015] The pharmaceutical composition comprises at least one MCP asdefined above, and optionally a pharmaceutically acceptable carrier orexipient. Examples of suitable pharmaceutical carriers are well known inthe art and include phosphate buffered saline solutions, water,emulsions, such as oil/water emulsions, various types of wetting agents,sterile solutions etc. Compositions comprising such carriers can beformulated by conventional methods. The pharmaceutical compositions canbe administered to the subject at a suitable dose. The dosage regimenmay be determined by the attending physician considering the conditionof the patient, the severity of the disease and other clinical factors.Administration of the suitable compositions may be effected by differentways, e.g. by intravenous, intraperetoneal, subcutaneous, intramuscular,topical or intradermal administration.

[0016] In a preferred embodiment, said MCP used in the methods and usesof the invention is selected from the group consisting of MCP-1, MCP-2,MCP-3, MCP4, MlP-1α RANTES, J-309 or any other CC-chemokine or classicalchemoattractants like N-farnesyl peptides, C5a, leukotriene B4 orPlatelet-activating factor (PAF)^(35,48).

[0017] In a particularly preferred embodiment, the method and uses ofthe invention are for the treatment of subjects suffering from occlusivedisease, preferably selected from the group consisting of coronaryartery diseases, cerebral occlusive diseases, peripheral occlusivediseases, visceral occlusive diseases, renal artery disease andmesenterial arterial insufficiency.

[0018] In a further preferred embodiment, the methods and uses of theinvention are for the treatment of subjects during or after exposure toan agent or radiation or surgical treatment which damage or destroyarteries.

[0019] In a preferred embodiment, the MCP used in the methods and usesof the invention is a recombinant MCP. DNA sequences encoding MCPs whichcan be used in the methods and uses of the invention are described inthe prior art, e.g., Garcia-Zepeda³⁴. Moreover, DNA and amino acidsequences of MCPs are available in the Gene Bank database. As describedabove, methods for the production of recombinant proteins are well-knownto the person skilled in the art; see, e.g., Sambrook, supra.

[0020] In a further preferred embodiment, the pharmaceutical compositionis designed for administration in conjugation with growth factors,preferably fibroblast growth factor or vascular endothelial growthfactor (VEGF). This embodiment is particularly suited for enhancing ofboth sprouting of capillaries (angiogenesis) and in situ enlargement ofpreexisting arteriolar connections into true collateral arteries.Pharmaceutical compositions comprising, for example, an MCP such asMCP-1, and a growth factor such as VEGF may be used for the treatment ofperipheral vascular diseases or coronary artery disease.

[0021] In another preferred embodiment, the method of the inventioncomprises

[0022] (a) obtaining cells from a subject;

[0023] (b) introducing a nucleic acid molecule encoding the MCP intosaid cells, thereby conferring expression and secretion of the MCP in aform suitable for the attraction of monocytes; and

[0024] (c) reintroducing the cells obtained in step (b) into thesubject.

[0025] It is envisaged by the present invention that the MCPs and thenucleic acid molecules encoding the MCPs are administered either aloneor in combination, and optionally together with a pharmaceuticallyacceptable carrier or exipient. Said nucleic acid molecules may bestably integrated into the genome of the cell or may be maintained in aform extrachromosomally. On the other hand, viral vectors may be usedfor transfecting certain cells or tissues, preferably cells and tissuesurrounding preexisting arteriolar connections. Elements capable oftargeting a nucleic acid molecule and/or protein to specific cells aredescribed in the prior art, for example Somia, Proc. Natl. Acad. Sci.,USA 92 (1995), 7570-7574. Thus, it is possible to employ the methods anduses of the invention for somatic gene therapy, which is based onintroducing of functional genes into cells by ex vivo or in vivotechniques and which is one of the most important applications of genetransfer; see, e.g., Schaper⁷³ and references cited therein.

[0026] Thus, in a preferred embodiment, the nucleic acid moleculecomprised in the pharmaceutical composition for the use of the inventionis designed for the expression and secretion of the MCP by cells in vivoin a form suitable for the attraction of monocytes by, for example,direct introduction of said nucleic acid molecule or introduction of aplasmid, a plasmid in liposomes, or a viral vector (e.g. adenoviral,retroviral) containing said nucleic acid molecule.

[0027] As discussed above, the growth of arteries from preexistingarteriolar connections is essential for the delivery of nutrition totumors. Thus, if the growth of said vessels to the tumor would besuppressed suppression and/or inhibition of tumor growth is to beexpected. Accordingly, the present invention also relates to a methodfor the treatment of tumors comprising contacting tissue or cells withan agent which suppresses the growth of collateral arteries and/or otherarteries from preexisting arteriolar connections through the attractionof monocytes. Agents which suppress the growth of collateral arteriesand/or other arteries from preexisting arteriolar connections may bepeptides, proteins, nucleic acids, antibodies, small organic compounds,hormones, neural transmitters, peptidomimics, or PNAs (Milner, NatureMedicine 1 (1995), 879-880; Hupp, Cell 83 (1995), 237-245; Gibbs, Cell79 (1994), 193-198).

[0028] The present invention further relates to the use of an agentwhich suppresses the growth of collateral arteries and/or other arteriesfrom preexisting arteriolar connections through the attraction ofmonocytes for the preparation of a pharmaceutical composition for thetreatment of tumors.

[0029] In a preferred embodiment, the agent used in the methods and usesof the invention as described above inhibits the biological activity ofa MCP and/or inhibits an intracellular signal triggered in the monocytesthrough the receptor for an MCP, preferably the aforementioned agentblocks and interaction of the MCP and its receptor. Various receptors ofMCPs are described in the prior art, for example in Charo⁶⁸ andChemokine Receptors⁶². Furthermore, it has recently been shown thatphosphorylation of the MCP-receptor mediates receptor desensitizationand internalization and that via altering the phosphorylation sites ofthe receptor the chemotactic response of leukocytes to MCP-1 and relatedchemokines can be modulated⁶⁷.

[0030] In another preferred embodiment, said receptor is selected fromthe group consisting of CCR1, CCR2, CCR4 and CCR5.

[0031] In a preferred embodiment, the agent which interaction of the MCPand its receptor is selected from the group consisting of

[0032] (i) an anti-MCP antibody and an anti-MCP-receptor antibody;and/or

[0033] (ii) a non-stimulatory form of an MCP protein and a soluble formof an MCP-receptor.

[0034] Anti-MCP or MCP-receptor antibodies can be prepared by well knownmethods using the purified MCP or its receptor or parts thereof as anantigen. Monoclonal antibodies can be prepared, for example, by thetechniques as described in Köhler and Milstein, Nature 256 (1975), 495,and Galfré, Meth. Enzymol. 73 (1981), 3, which comprise the fusion ofmouse myeloma cells to spleen cells derived from immunized mammals.Furthermore, antibodies or fragments thereof to the aforementioned MCPsor their receptors can be obtained by using methods which are described,e.g., in Harlow and Lane “Antibodies, A Laboratory Manual”, CSH Press,Cold Spring Harbour, 1988. These antibodies may be monoclonalantibodies, polyclonal antibodies or synthetic antibodies as well asfragments of antibodies, such as Fab, Fv, or scFv fragments etc.

[0035] Non-stimulatory forms of MCPs and antagonists of MCP-receptorshave been described, for example, in Gong⁶⁵.

[0036] In another embodiment, the agent which suppresses the growth ofcollateral arteries and/or arteriogenesis is an anti-sense RNA of theMCP or of its receptor. It might be desirable to inactivate theexpression of the gene encoding the MCP and/or encoding its receptor.This can be achieved by using, for example, nucleic acid molecules whichrepresent or comprise the complementary strand of the mRNA transcript orpart thereof encoding the MCP or its receptor. Such molecules may eitherbe DNA or RNA or a hybrid thereof. Furthermore, said nucleic acidmolecule may contain, for example, thioester bonds and/or nucleotidesanalogues, commonly used in oligonucleotide anti-sense approaches. Saidmodifications may be useful for the stabilization of the nucleic acidmolecule against endo- and/or exonucleases in the cell. Said nucleicacid molecules may also be transcribed by an appropriate vectorcontaining a chimeric gene which allows for the transcription of saidnucleic acid molecule in the cell. Such nucleic acid molecules mayfurther contain ribozyme sequences which specifically cleave the mRNAencoding the MCP or its receptor. Furthermore, oligonucleotides can bedesigned which are complementary to a region of the gene encoding theMCP or its receptor (triple helix; see Lee Nucl. Acids Res. 6 (1979),3073; Cooney, Science 241 (1988), 456 and Dervan, Science 251 (1991),1360), thereby preventing transcription and the production of the MCP orits receptor.

[0037] In a preferred embodiment, the anti-sense RNA is designed to beexpressed in vascular cells or cells surrounding preexisting arteriolarconnections to a tumor.

[0038] In a preferred embodiment, methods and uses of the invention areemployed for the treatment of a tumor which is a vascular tumor,preferably selected from the group consisting of Colon Carcinoma,Sarcoma, Carcinoma in the breast, Carcinoma in the head/neck,Mesothelioma, Glioblastoma, Lymphoma and Meningeoma.

[0039] In a preferred embodiment, the pharmaceutical composition in theuse of the invention is designed for administration by catheterintraarterial, intravenous, intraperitoneal or subcutenous routes. Inthe examples of the present invention the human form of the MCP-1protein was administered locally via osmotic minipump. Positiveimmunohistochemical staining for BrdU infused into two animals via thesame route as MCP-1 demonstrated that local delivery of substances intothe collateral circulation is feasible.

[0040] The said MCP and its encoding nucleic acid molecule may be usedfor therapeutical purposes in various forms. Either as in theexperiments described herein, locally via implanted pumps, or asarterial or venous boluses either systemically or locally via speciallydesigned catheters or other device. They may also be injectedintramuscularly or into any other tissues in which collateral arterygrowth needs to be promoted. Alternatively they can be bound tomicrocapsules or microspheres before injection.

[0041] Another approach would be to use a gene-transfer approach, eitherusing a plasmid, or a plasmid embedded in liposomes, or viral vectors.One may either use an in vivo gene-transfer approach for which multipledevices, like double balloon or other catheters have been designed orvia direct injection into the targeted tissue as described above.Alternatively it is possible to use an ex vivo approach isolating cellswhich are known to lodge in tissues in which vessel growth needs to bepromoted or inhibited from the body which are then transfected using oneof the above mentioned methods and reinjected.

[0042] The use and methods of the invention can be used for thetreatment of all kinds of diseases hitherto unknown as being related toor dependent on the modulation of the growth of collateral arteriesand/or other arteries from preexisting arteriolar connections. Themethods and uses of the present invention may be desirably employed inhumans, although animal treatment is also encompassed by the methods anduses described herein.

THE FIGURES SHOW

[0043]FIG. 1: Monocyte/macrophage accumulation after femoral arteryocclusion in the rabbit hindlimb. A) A-monocyte adheres to the wall ofan excised collateral artery (arrow); two other macrophages staininggreen (bright) have already penetrated the vessel wall. B) Macrophagesare also found interstitially in the lower limb (arrows). C) and D)Monocytes/macrophages staining green (bright) are much more numerous inanimals treated with MCP 1 (scale bars: 20 mm).

[0044]FIG. 2: Post-mortem angiograms of rabbit hindlimbs after one weekof femoral artery occlusion. A) Without MCP-1 treatment. B) After oneweek of local MCP-1 infusion. The density of collateral vessels withtypical corkscrew appearance is markedly increased in hindlimbs ofanimals treated with MCP-1.

[0045]FIG. 3 A) Staining of bromodeoxyuridine (BrdU) (green (bright)fluorescence) infused continuously by minipump as proliferation markerand counterstained with phalloidin-TRITC as marker for actin: Pronouncedincorporation of BrdU in endothelial and smooth muscle cells during thefirst week of femoral artery occlusion. B) Specific staining ofcapillaries with an antibody against CD 31 in a normal gastrocnemialmuscle. C) The same muscle stained for CD 31 after one week ofocclusion; the number of capillaries has increased. D) Gastrocnemialmuscle after one week of occlusion and MCP 1 infusion; capillaries aremore numerous after MCP 1 treatment (Scale bars in all pictures: 20mm).

[0046]FIG. 4: Bulk conductance of rabbit hindlimbs after one week offemoral artery occlusion with local MCP-1 infusion in comparison tocontrol hindlimbs after acute, one week, 3 weeks or no occlusion. Bulkconductance in animals treated with MCP-1 was significantly higher thanin control animals after the same time of femoral artery occlusion andreached values of non occluded legs (*p<0.05 and**p<0.01 as compared toacute occlusion;

p<0.05 as compared to one week of occlusion without MCP-1 treatment).

[0047]FIG. 5: Collateral conductance of rabbit hindlimbs after one weekof femoral artery occlusion with local MCP-1 infusion in comparison tocontrol hindlimbs after acute, one week, and 3 weeks of occlusion indifferent regions. Collateral conductance in animals treated with MCP-1was significantly higher than in control animals after the same time offemoral artery occlusion in the quadriceps and adductor longus muscleregion. These values tended to be higher than those observed in controlanimals after three weeks of femoral arterv occlusion (*p<0.05and**p<0.01 as compared to acute occlusion;

p<0.01 as compared to one week of occlusion without MCP-1 treatment).

[0048]FIG. 6: Peripheral conductance of rabbit hindlimbs after one weekof femoral artery occlusion with local MCP-1 infusion in comparison tocontrol hindlimbs after acute, one week and 3 weeks of occlusion.Peripheral conductance in animals treated with MCP-1 was significantlyhigher than in control animals after the same time of femoral arteryocclusion. Similar to collateral conductance these values tended to behigher than those observed in control animals after three weeks offemoral artery occlusion (*p<0.05 and**p<0.01 as compared to acuteocclusion;

p<0.01 as compared to one week of occlusion without MCP-1 treatment).

[0049]FIG. 7: Number of collateral arteries identified by their stemregions, midzone regions and reentry regions in stereoscopic,3-dimensional angiograms. The number of collateral arteries after oneweek of occlusion (right leg) was almost twice as high in animal treatedwith MCP-1 as compared to animals treated with the carrier alone. Nosignificant differences were found in the non-occluded left control leg.

THE EXAMPLES ILLUSTRATE THE INVENTION EXAMPLE 1

[0050] Femoral Artery Occlusion of Animals and Local Delivery of Agents

[0051] The present study was performed with permission of the State ofHesse, Regierungspräsidium Darmstadt, according to § 8 of the German Lawfor the Protection of Animals. It conforms with the Guide for the Careand Use of Laboratory Animals published by the US National Institutes ofHealth (NIH Publication No.85-23, revised 1985). Twelve rabbits weresubjected to 7 days of bilateral femoral artery occlusion. They wererandomly assigned to either receive Monocyte Chemotactic Protein-1(MCP-1; PeproTech Inc, Rocky Hill, N.J., USA) locally via osmoticminipump (2ML-2 Alza Corp, USA; 3 mg in 2 ml phosphate buffered saline(PBS) ata rate of 10 ml/h), PBS via osmotic minipump or no treatment.Nine additional animals were subjected to either no, acute or 21 days offemoral artery occlusion for comparison.

[0052] Two animals were supplied with an osmotic minipump (2ML-2 AlzaCorp, USA) delivering bromodeoxyuridin (BrdU: Sigma Chemicals, St.Louis) via the same route as MCP-1 to verify the function of the localdelivery system and to study the proliferation of collateral arteriesand capillaries.

[0053] For the initial surgery the animals were anesthetized with anintramuscular injection of ketamin hydrochloride (4-8 mg per kilogrambody weight) and xylazin (8-9 mg per kilogram body weight).Supplementary doses of anesthetic (10-20 % of the initial dose) weregiven intravenously as needed. Surgery was performed under sterileconditions. Femoral arteries were exposed and cannulated with a sterilepolyethylene catheter (1 mm i.d., 1.5 mm o.d.) pointing upstream withthe tip of the catheter positioned distally of the branching of thearteria circumflexa femoris. The catheter itself was connected to theosmotic minipump (2ML-2 Alza Corp, USA) which was implanted under theskin of the lower abdomen. Rabbits were outfitted with a speciallydesigned body suit which allowed them to move freely but preventedselfmutilation. They were housed together in a large cage with freeaccess to water and chow to secure mobility. Before sacrifice theanimals received another intramuscular injection of ketaminhydrochloride and xylazin. The animals then underwent tracheostomy andwere artificially ventilated. Anesthesia was deepened with pentobarbital(12 mg/Kg bodyweight per hour). The carotid artery was cannulated forcontinuous pressure monitoring. The arteria saphena magna (anteriortibial artery in humans; main arterial supply to the lower limb and footin the rabbit) was exposed just above the ankle and cannulated withpolyethylene tubing (0.58 mm i.d., 0.96 mm o.d.). They were connected toa Statham P23DC pressure transducer (Statham, Spectramed, USA) formeasurement of peripheral pressures. After heparinization with 5000units of heparin both external iliac arteries were exposed andcannulated with 2.0 mm bore metal tubing. The abdominal circumflexartery and the arteria spermatica were ligated and a tourniquet placedproximally around both thighs leaving the femoral artery patent. Thefemoral and sciatic vein were incised for drainage of venous blood. Theanimals then were bled, the legs were amputated above the hip andquickly transferred to the perfusion apparatus. No animal was lostduring or after the primary operation. It was also not observed anygangrene or gross impairment of function after femoral artery occlusion.Two animals had to be excluded from the study because of air embolism.After finishing the experiment all fluid remaining in the reservoir ofthe minipump was collected and weighed. In the two control animalsreceiving bromodesoxyuridine (BrdU), BrdU staining was performed bystandard immunohistochemical methods described elsewhere²⁶. Evaluationof fluids remaining in the reservoir revealed that pumping at a rate of10 ml/h was accomplished in all experiments. Positiveimmunohistochemical staining for BrdU demonstrated that local infusioninto the collateral circulation via osmotic minipump was feasible.

EXAMPLE 2

[0054] Ex Vivo Pressure-Flow Relations

[0055] The legs were perfused with autologuous oxygenated blood warmedto 37° C. using a Stoeckert roller pump (Stoeckert GmbH, Germany) and aJostra M2 membrane oxygenator (Jostra GmbH, Germany). Hematocrit waskept between 34 % and 37 % and oxygen saturation at 99 %. Maximalvasodilation was achieved by adding 25 mg of papaverine (SigmaChemicals, St. Louis USA) to the perfusate (priming volume: 60 ml). Thelegs were perfused at three different pressure levels (40, 60 and 80mmHg). After stabilization radioactive microspheres were injected and areference sample drawn using a syringe pump (Braun Melsung, Germany).For each pressure level microspheres labeled either with Ruthenium,Cerium and Niobium or Scandium (Dupond NEN Products, USA) were randomlychosen. This allowed to relate tissue perfusion to different perfusionpressures. Total flow was determined using an ultrasonic inline flowprobe connected to a T201 flowmeter (Transonic Systems, Inc, USA).Systemic pressures and peripheral capillary pressures were traced with aStatham P23DC pressure transducer (Statham, Spectramed, USA). Allrecordings were transferred online to a computerized recording system(MacLab, Apple Microsoft USA) from which they were recovered for furtherprocessing. Quadriceps, adductor lonaus and adductor magnus,gastrocemius, soleus and peroneal musces were dissected from the leg andeach muscle was divided into five consecutive samples from the proximalto the distal end. Samples were weighed and subsequently analyzedtogether with the respec-tive reference samples using a Ge-detector asdescribed previously²⁷. Of the total 27 hindlimbs which were perfused 4were excluded because peripheral presssures could not be obtained and 1was excluded from the determination of collateral and capillaryconductances because of sampling errors. There were no significantdifferences in conductances between animals receiving PBS via minipumpand animals receiving no treatment (bulk conductance: 57.2±8.60 vs.69.2±10.01; collateral conductance: 24.5±5.69 vs. 25.3±3.29; all data inml/min/100 mmHg). Therefore these two groups were combined in the finalanalysis.

[0056] For the calculation of sample flows mean sample activity per gramof muscle weight (Am/g) was used and related to total flow per gram ofmuscle weight (F_(t)/g) which allowed the calculation of sample flow(F_(s)) using the equation F_(s)=F_(t)/A_(m)×A_(s). This correlated wellwith the calculation of sample flow (F_(s)) from sample activity(A_(s)), reference sample activity (A_(r)), weight of the referencesample (W_(r)) and time of reference sample withdrawal (t) following theequation F_(s)=A_(s)/A_(r)×W_(r)/t.

[0057] In the present model collateral arteries developing after femoralartery occlusion in typical corkscrew formation supply blood to thedistal adductor region and the lower leg. Systemic pressure (SP) andperipheral pressure was used in the saphenous artery (PP). Venouspressure was equal to atmospheric pressure (AP; zero in the presentcase). Since arterial resistances are much lower than collateral andperipheral resistances they can be neglected. SP represents the pressureat the stem region of the collateral arteries. PP is the pressure at thereenty region and is identical to the pressure head of the circulationin the lower leg, AP the pressure at the venous end of the peripheralcirculation. Collateral flow (Fc) is equal to the sum of flow to thetissue of the distal adductor (FdTA) plus the flow to the tissue of thelower leg (FTII). (Flow to the bone was very small and the main arterialsupply to the foot was ligated. Therefore these values were neglected inour calculation). Collateral resistance (Rc) was defined as pressuredifference between perfusion pressure (SP) and peripheral pressure (PP)divided by the flow going to the distal adductor and the lower leg.Peripheral resistance (R_(p)) was defined as peripheral pressure (PP)divided by flow to the lower leg (FTII) and bulk conductance was definedas systemic pressure (SP) divided by bulk flow recorded with theultrasonic flow probe. The reciprocal values of these resistancesrepresent collateral-, peripheral- and bulk conductance (Cc, Cp and Cb)Because a positive pressure intercept is observed even at maximalvasodilation all conductances were calculated from the slope of pressureflow relations.

[0058] After one week of femoral artery occlusion bulk conductance ascalculated from pressure flow relations was significantly higher inanimals treated with MCP-1 (142.1±31.71 ml/min/100mmHg versus 66.2±7.76ml/min/ 100mmHg; p<0.05)(FIG. 4). After seven days of occlusion bulkconductances of MCP-1 treated animals reached levels even higher than inuntreated animals after three weeks of femoral artery occlusion and wascomparable to values in non-occluded hindlimbs.

[0059] Collateral conductance also was significantly higher after oneweek of occlusion in animals treated with MCP1 as compared to animalswithout this treatment ( 70.6±19.23 ml/min/100mmHg versus 25.1±2.59ml/min/100mmHg; p<0.01)(FIG. 5). Collateral conductance of animals thathad received MCP-1 for one week tended to be even larger than inuntreated animals after three weeks of femoral artery occlusion in allareas in which collateral growth was observed. Conductance in the calfalso was significantly higher after one week of femoral artery occlusionin animals with MCP-1 treatment as compared to rabbits which had notreceived MCP-1 (119.3±22.37 ml/min/100mmHg versus 45.4±6.80ml/min/100mmHg; p<0.01) (FIG. 6). All data are presented as mean ± SEM.Intergroup comparisons were performed by unpaired Student's t-test. Inthe case of unequal variances the Mann-Whitney Rank Sum test was used.Probability values of 0.05 or less were required for assumption ofstatistical significance.

[0060] Treatment with MCP-1 increased both collateral and peripheralconductance 2-fold as compared to untreated animals after 7 days offemoral artery occlusion. Thus animals locally injected with MCP-1reached normal conductance values after one week of occlusion whereasconductance values in untreated animals did not return to normal levelseven three weeks after occlusion. As mentioned above MCP-1 is mainlyknown as chemoattractant for monocytes^(31,35). One possible explanationwould therefore be that MCP-1 exerts its pronounced effects oncollateral- and peripheral conductance via attraction and activation ofmonocytes that in turn produce growth factors which lead to theproliferation of endothelial and smooth muscle cells. This requires thatmonocytes adhere to the small arteriolar connections which are verylikely the origin of our collateral arteries^(35,48,49). Thesepreexisting arteriolar connections experience a large increase in shearstress when the main arterial supply to the lower leg is occluded.

EXAMPLE 3

[0061] Post Mortem Angiography

[0062] After maximal vasodilatation legs were warmed to 37° C. andperfused with Krebs-Henseleit buffered saline for one minute followed byperfusion with contrast medium based on bismuth and gelatine accordingto a formula developed by Fulton²⁸. Subsequently the contrast medium wasallowed to gel by placing the limb on crushed ice and angiograms weretaken at two different angles in a Balteau radiography apparatus(Machlett laboratories, USA) using a single enveloped Structurix D7 DWfilm (AGVA, Germany). The resulting stereoscopic pictures allowedanalysis of collateral growth in three dimensions.

[0063] Post mortem angiograms exhibited corkscrew collaterals mainly inthe adductor longus, adductor magnus and vastus intermedius musclesconnecting the perfusion bed of the arteria femoralis profunda to thatof the arteria saphena parva in the adductor muscles and the perfusionbed of the arteria circumflexa femoris lateralis to that of the arteriaegenuales in the quadriceps muscle. Angiograms taken from hindlimbs ofanimals with MCP-1 treatment showed a remarkable increase in the densityof these collateral vessels (FIG. 2 A and B). No collateral vessels werevisible on angiograms in the lower limb of normal and MCP-1 treatedanimals.

EXAMPLE 4

[0064] Histological Studies

[0065] The abdominal aorta was cannulated with a 2 mm bore metalcannula, the chest was opened and the heart exposed. After incision ofthe right atrium to allow drainage of rinsing solution and fixativeperfusion was started with a rinsing solution containing 0.5% BSA, 5 mMEDTA, 0.317 mg/l Adenosin in phosphate buffered saline (PBS)×1.5 for 5min followed by fixation with formalin 4% in the rinsing solutionwithout BSA for 20 min. Subsequently a post mortem angiography wasperformed as described in Example 3. This allowed the preciselocalization and excision of collateral vessels, their stem and reentryregions.

[0066] For immunohistological studies, samples were kept in 20 %saccharose overnight and then frozen and mounted on cork in nitrogencooled methylbutane at −130° C. They were stored at −80° C. untilfurther processing. For visualization of BrdU cryostat sections of 20 mmwere obtained in a Leica CM 3000 cryotom, mounted onto silicone coatedslides and incubated in 2mol/l HCl at 38° C. for 20 minutes. Afterrinsing in PBS 3 times for 5 minutes they were incubated with theprimary antibody against BrdU (Clone BU20a, DAKO Corp.), 1:20 in PBS at4° C. overnight. For detection the samples were incubated with abiotinylated donkey antimouse antibody (DIANOVA Corp) 1:100 in PBS forone hour followed by incubation with streptavidin-cy2 (Biotrend, Koeln,Germany) 1:100 in PBS for 30 minutes. Finally sections werecounterstained either with 7-aminoactinomycin D (7-MD 1:50 in PBS,Molecular Probes, Eugene, Oreg. USA) as nuclear stain or phalloidin-TRTC(1:100 in PBS) as marker for actin. Slides were mounted in Mowiol(Hoechst, Frankfurt/M, Germany) and viewed by Leica confocal lasermicroscope. Neighboring sections treated identically but omitting theprimary antibody served as a negative control. Immunohistochemicalstaining of capillary endothelial cells was performed following theprotocol described above but with an antibody against CD 31 (DACO,Germany), an endothelial specific antigen, as primary antibody. Stainingfor macrophages was performed using RAM 11 (DACO, Germany), a specificantibody against rabbit macrophages as primary antibody. After femoralartery occlusion monocytes/macrophages were found to accumulate invessel walls of excised collateral arteries and intersitially in thelower limb (FIG. 1A and B). They were more numerous in animals treatedwith MCP-1 (FIG. 1C and D). Furthermore white plaques were seenmacrospically around the infusion site in all animals receiving MCP-1.These plaques contained large numbers of mononuclear cells whichpredominantly were identified as monocytes/macrophages byimmunohistochemical staining with Ram 11 (Dako GmbH, Hamburg, Germany).By macroscopical inspection of the injection site and histologicalexamination of collateral arteries from the thigh and tissue sectionsfrom calf muscles it became evident that MCP-1 injection had led to anincrease of monocyte accumulation in our experiment. Positive stainingof excised collateral arteries for BrdU provided evidence thatcollateral vessels observed on angiography in the thigh were trulyproliferating.

[0067] Collateral arteries excised after 7 days of occlusion showedproliferation of endothelial- and smooth muscle cells on BrdU staining(FIG. 3 A). Proliferation of capillary endothelial cells was seen in thelower limb leading to an increase in the number of capillaries 7 daysafter occlusion (Control leg: FIG. 3 B; leg after 7 days of occlusion:FIG. 3 C). MCP 1 treated animals showed more capillaries in the lowerlimb than untreated animals after a week of occlusion indicatingenhancement of capillary sprouting by MCP 1 (FIG. 3 D).

[0068] The immunohistochemical studies after continuous BrdU infusionclearly demonstrated that collateral vessel formation in the thighinvolved proliferation of endothelial- and smooth muscle cells, giventhe fact that the normal generation time for endothelial cells andsimilar for smooth muscle cells is at least six months and proliferationis usually not seen in normal arteries³⁹. The degree of proliferation issimilar to that of collateral arteries in the dog heart after ameroidconstrictor placement and approaches that of tumors⁴⁰. Although thisdoes not exclude the possibility that MCP-1 enhances collateral arteryproliferation via hypothetical, unrecognized chronic vasodilatoryeffects, the rapidity and magnitude of the increase in collateralconductance is far higher than with any other known vasodilator⁴¹⁻⁴⁴.Furthermore monocytes have been shown to downregulate nitric oxidesynthase, a very potent vasodilator, in cultured aortic endothelialcells suggesting that MCP-1 would rather inhibit than enhancevasodilation⁴⁵. Therefore vasodilation is a very unlikely explanationfor the above findings. The higher density of collateral arteries on theangiograms further supports the notion that collateral artery growth isresponsible for the increase in collateral conductance.

[0069] In contrast to the thigh were the density of collateral arteriesincreased, more capillaries were found in histological sections fromcalf muscles of MCP-1 treated animals as compared to control animalsafter seven days of occlusion. An antibody against CD31 (PECAM) waschosen as marker for endothelial cells because this cell adhesionmolecule is constitutively expressed on all endothelial cells and notdependent on their phenotype or activation^(46,47). Using BrdU as amarker for proliferation only proliferating capillaries in the calfmuscles were detected. No other vessel type was found to grow in thisregion. As for collateral conductance passive vessel enlargement due tovasodilation can be excluded as a reason for peripheral conductancechanges by performing the measurements at maximal vasodilatation. Thuschanges in peripheral conductance are most likely attributable tocapillary sprouting.

[0070] The histological data suggests that more monocytes accumulate inMCP-1 treated animals. Since monocytes are potentially capable ofproducing large amounts of growth factors this further supports thehypothesis that monocytes are the mediator of the changes seen withMCP-1 treatment.

[0071] In summary, our results have shown that local infusion of MCP-1,a potent and specific chemoattractant for monocytes, is able to markedlyincrease collateral- as well as peripheral conductance. Angiographic andhistological findings indicate that this effect is due to augmentedcollateral artery- and capillary proliferation and suggest thatadhesion, activation and migration of monocytes play an important rolein both types of vessel growth.

EXAMPLE 5

[0072] Number of Collateral Arteries

[0073] Post-mortem angiographies were obtained as described in Example3. For quantification the bone was extracted and the thigh muscles wereunfolded before placing the tissue in the Balteau radiography apparatus.This allowed the identification and counting of individual collateralarteries by virtue of their stem regions, midzone regions and reentryregions on stereoscopic angiograms. The number of collateral arteriesthus counted did not differ in individual animals when obtainedindependently by four different observers. Angiograms were obtained fromsix animals receiving MCP-1 locally via osmotic minipump afterunilateral femoral artery occlusion and compared to angiograms of sixanimals receiving the carrier PBS via the same route after femoralartery occlusion. The results are shown in FIG. 7.

[0074] After seven days of occlusion the number of collateral arterieswas almost twice as high in animals receiving MCP-1 as compared toanimals receiving PBS alone (30.17±1.96 vs 16.17±1.4; P<0.001); seeTable 1. TABLE 1 PBS l. 5.00 ± 0.45 MCP-1 l. 6.67 ± 1.17 PBS r. 16.17 ±1.40 MCP-1 r. 30.17 ± 1.96 PBS r. vs. MCP-1 r. P = 0.0002

[0075] Table 1: Number of collateral arteries identified by their stemregions, midzone regions and reentry regions in stereoscopic,3-dimensional angiograms. The number of collateral arteries after oneweek of occlusion (right leg) was almost twice as high in animal treatedwith MCP-1 as compared to animals treated with the carrier alone. Nosignificant differences were found in the non-occluded left controllegs. There was no difference in the number of collateral arteriesbetween MCP-1—and carrier-treated animals in the non-occluded controllegs suggesting that additional mechanisms are necessary in order topromote collateral growth which are elicited by occlusions. MCP-1therefore will not enhance collateral growth or growth of other vesselsin sites without vessel occlusions.

EXAMPLE 6:

[0076] Long-Term Effects of MCP-1 Treatment as seen by MRI Scanning

[0077] Six animals treated with MCP-1 and 6 control animals wereinvestigated with MRI-scanning acutely after 7 days, 2 weeks, 1, 2 and 3months after unilateral femoral artery occlusion. The anatomicalstructure was analyzed with highresolution T1-SE Images. MR-angiographywas performed with a 3-D FISP Sequence. Perfusion was measured after anintravenous bolus of GD-DTPA with a TFL-SR-Sequence. Analysis wasperformed according to the different muscle groups. The number ofcollateral arteries in MCP-1 treated animals was higher throughout theinvestigated time frame. In contrast to control animals retrogradefiling of the femoral artery was already normalized after 2 weeks ofocclusion in MCP-1 treated animals.

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1. A method for enhancing the growth of collateral arteries and/or otherarteries from preexisting arteriolar connections comprising contactingtissue or cells with a monocyte chemotactic protein (MCP) or a nucleicacid molecule encoding said MCP.
 2. Use of a monocyte chemotacticprotein (MCP) or a nucleic acid molecule encoding said MCP for thepreparation of a pharmaceutical composition for enhancing collateralgrowth of collateral arteries and/or other arteries from preexistingarteriolar connections.
 3. The method of claim 1 or the use of claim 2,wherein said MCP is selected from the group consisting of MCP-1, MCP-2,MCP-3, MCP4, MIP-1α, RANTES, J-309 or any other CC-chemokine orclassical chemoattractants like N-farnesyl peptides, C5a, leukotriene B4or Platelet-activating factor (PAF).
 4. The method of claim 1 or 3 orthe use of claim 2 or 3 for the treatment of subjects suffering fromocclusive diseases.
 5. The method or the use of claim 4, wherein theocclusive disease is an arterial occlusive disease selected from thegroup consisting of coronary artery diseases, cerebral occlusivediseases, peripheral occlusive diseases, visceral occlusive diseases,renal artery diseases and mesenterial arterial insufficiency.
 6. Themethod of claim 1 or 3 or the use of claim 2 or 3 for the treatment ofsubjects during or after exposure to an agent or radiation or surgicaltreatment which damage or destroy arteries.
 7. The method of any one ofclaims 1 or 3 to 6 or the use of any one of claims 2 to 6, wherein theMCP is a recombinant MCP.
 8. The use of any one of claims 2 to 7,wherein the pharmaceutical composition is designed for administration inconjugation with growth factors like fibroblast growth factor orvascular endothelial growth factor.
 9. The method of any one of claims 1or 3 to 7, comprising (a) obtaining cells from a subject; (b)introducing a nucleic acid molecule encoding the MCP into said cells,thereby conferring expression and secretion of the MCP in a formsuitable for the attraction of monocytes; and (c) reintroducing thecells obtained in step (b) into the subject.
 10. The use of any one ofclaims 2 to 8, wherein the nucleic acid molecule in the pharmaceuticalcomposition is designed for the expression and secretion of the MCP bycells in vivo in a form suitable for the attraction of monocytes.
 11. Amethod for the treatment of tumors comprising contacting tissue or cellswith an agent which suppresses the growth of collateral arteries and/orother arteries from preexisting arteriolar connections through theattraction of monocytes.
 12. Use of an agent which suppresses the growthof collateral arteries and/or other arteries from preexisting arteriolarconnections through the attraction of monocytes for the preparation of apharmaceutical composition for the treatment of tumors.
 13. The methodof claim 11 or the use of claim 12 wherein the agent inhibits thebiological activity of a MCP and/or inhibits an intracellular signaltriggered in the monocytes through the receptor for a MCP.
 14. Themethod or the use of claim 13, wherein the agent blocks an interactionof the MCP and its receptor.
 15. The method or the use of claim 14,wherein the receptor is selected from the group consisting of CCR1,CCR2, CCR4 and CCR5.
 16. The method or the use of claim 14 or 15,wherein the agent which blocks an interaction of the MCP and itsreceptor is selected from the group consisting of (i) an anti-MCPantibody and an anti-MCP-receptor antibody; and/or (ii) anon-stimulatory form of an MCP protein and a soluble form of anMCP-receptor.
 17. The method of claim 11 or the use of claim 12, whereinthe agent is an antisense RNA of the MCP or of its receptor.
 18. Themethod or the use of claim 17, wherein the antisense RNA is designed tobe expressed in vascular cells or cells surrounding preexistingarteriolar connections to a tumor.
 19. The method of any one of claims11 or 13 to 18 or the use of any one of claims 12 to 18, wherein thetumor is a vascular tumor.
 20. The method or the use claim 19, whereinthe tumor is selected form the group consisting of Colon Carcinoma,Sarcoma, Carcinoma in the breast, Carcinoma in the head/neck,Mesothelioma, Glioblastoma, Lymphoma and Meningeoma.
 21. The use of anyone of claims 2 to 8 or 12 to 20, wherein the pharmaceutical compositionis designed for administration by catheter intraarterial, intravenous,intraperitoneal or subcutenous routes.